Controlling the onset and progression of mitotic cell division is critical to prevent human cancer. Much of this regulation involves the timely activation and inactivation of cyclin-dependent protein kinases that regulate progression through the cell cycle. In addition, cells must ensure that their genetic information is accurately transmitted to each daughter cell by activating a series of DNA damage-response kinases to halt the cell cycle in response to genotoxic stress. Many details of the molecular events that occur downstream of mitotic and DNA damage-response kinases, however, are unknown. In this regard, phosphoSerine/Threonine (pSer/pThr)-binding domains such as 14-3-3 proteins, FHA domains, WW domains, and WD40 repeats are increasingly emerging as critical doma ns that link upstream kinase signaling cascades to downstream effector functions by directly mediating the phosphorylation-dependent formation of multi-protein signaling complexes. The long-term goals of the applicant are to identify and characterize (pSer/pThr)-binding domains involved in regulating cell proliferation. In this proposal we seek funds to develop and improve a new phosphopeptide library-based proteomic screening technology that simultaneously: (1) reveals specific pSer/pThr-binding domains downstream of cyclin-dependent kinases and DNA damage-responsive kinases; (2) allows determination of the optimal sequence motifs recognized by the newly-identified domain; (3) provides reagents for biophysical, cell biological, and structural studies of the function of the newly identified domain in regulating mitotic progression, as well as reagents for high-throughput screening for discovery of small molecule inhibitors; and (4) facilitates bioinformatics and systems biology efforts to identify specific kinase targets within signaling pathways that mediate cell cycle control. This technology will have a major impact on proteomicsand bioinformatics/systems-biology based approaches to define kinase-dependent molecular pathways in cancer biology and will identify new therapeutic targets for drug design.